In many cases, early detection of disease improves outcomes; as a result, search for molecular biomarkers became an important direction in biomedical research. Ideally, disease-specific biomarkers should be sensitive, specific, inexpensive, versatile, rapid, and observer-independent to allow objective screening of asymptomatic people. In addition, it would be advantageous to have a uniform platform for development of biomarkers for multiple diseases. Changes in DNA methylation are found in different diseases and can be detected in circulating cell-free DNA from blood; however, there is only a certain probability of methylation for each specific site. To solve this problem and increase the accuracy of detection, a composite biomarker with multiple informative elements can be used. While the multitude of methylation sites indicates that multiple potential components exit for each biomarker, selection of informative components is difficult and requires a platform for simultaneous measurements of methylation in multiple sites of each clinical sample. Such a tool will be developed in this project (Aim 1) and its performance will be tested by selecting informative elements for a breast cancer biomarker (Aim 2). The proposed tool consists of a customized array for methylation detection in promoters of genes that are known to be methylated in different diseases; a procedure for isolating cell-free DNA from small amounts of plasma; and a technique for methylation detection in this DNA. The approach was tested with a 56 gene prototype; accuracy of detection was 70-90% for different diseases. The proposed array will contain the majority of abnormally methylated genes that can be analyzed by this technique in order to select additional highly informative genes and increase the accuracy. To confirm the utility of the tool, biomarkers for detection of invasive breast cancer and ductal carcinoma in situ will be developed. As a result, biomarker components for one of the most frequent cancers in females will be identified as well. Once the problem of finding informative elements is solved, only they will be measured in a dedicated screening assay for breast cancer. It is likely that the tool will find application for biomarker research in different clinical areas - e.g. in oncology, psychiatry, prenatal diagnostics, and pharmacology - and in other areas of biology, e.g. in embryology and in stem cell research, where methylation is involved in imprinting and embryonic programming. Public Health Relevance Statement: In this project a platform for testing DNA methylation in multiple sites will be developed and validated with DNA from blood of healthy women and breast cancer patients. The developed platform will allow selection of blood-based biomarkers for different diseases, which will translate into a rapid, inexpensive, and very accurate test for their detection.